Lesley Robson

Wnt signalling regulates myogenic differentiation in the developing avian wing

The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and ΔLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.

Craniofacial development: the tissue and molecular interactions that control development of the head

The molecular cascades that control craniofacial development have until recently been little understood. The paucity of data that exists has in part been due to the complexity of the head, which is the most intricate regions of the body. However, the generation of mouse mutants and the identification of gene mutations that cause human craniofacial syndromes, together with classical embryological approaches in other species, have given significant insight into how the head develops. These studies have emphasized how unique the head actually is, with each individual part governed by a distinct set of signalling interactions, again demonstrating the complexity of this region of the body. This review discussed the tissue and molecular interactions that control each region of the head. The processes that control neural tube closure together with correct development of the skull, midline patterning, neural crest generation and migration, outgrowth, patterning, and differentiation of the facial primordia and the branchial arches are thus discussed. Defects in these processes result in a number of human syndromes such as exencephaly, holoprosencephaly, musculoskeletal dysplasias, first arch syndromes such as Riegers and Treacher-Collins syndrome, and neural crest dysplasias such as DiGeorge syndrome. Our current knowledge of the genes responsible for these human syndromes together with how the head develops, is rapidly advancing so that we will soon understand the complex set of molecular and tissue interactions that build a head.

Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex(®), a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex(®) fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex(®) fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex(®) fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex(®) conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex(®) conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex(®) conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.

Omega-3 polyunsaturated fatty acids increase the neurite outgrowth of rat sensory neurones throughout development and in aged animals

Polyunsaturated fatty acids (PUFA) of the omega-3 series and omega-6 series modulate neurite outgrowth in immature neurones. However, it has not been determined if their neurotrophic effects persist in adult and aged tissue. We prepared cultures of primary sensory neurones from male and female rat dorsal root ganglia (DRG), isolated at different ages: post-natal day 3 (P3) and day 9 (P9), adult (2-4 months) and aged (18-20 months). Cultures were incubated with the omega-6 PUFA arachidonic acid (AA) and the omega-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), at 0.8, 4, 8 and 40muM. PUFA increased neurite outgrowth throughout the developmental stages studied. The effects of omega-3 PUFA, in particular DHA, were still prominent in aged tissue. The amplitude of the effects was comparable to that of nerve growth factor (NGF; 50ng/ml) and all-trans-retinoic acid (ATRA; 0.1muM). The effects of PUFA were similar in cells positive or negative for the N52 neurofilament marker. Our results show that omega-3 PUFA have a marked neurite-promoting potential in neurones from adult and aged animals.

Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm

The T‐box transcription factor Tbx1 has been implicated in DiGeorge syndrome, the most frequent syndrome due to a chromosomal deletion. Gene inactivation of Tbx1 in mice results in craniofacial and branchial arch defects, including myogenic defects in the first and second branchial arches. A T‐box binding site has been identified in the Xenopus Myf5 promoter, and in other species, T‐box genes have been implicated in myogenic fate. Here we analyze Tbx1 expression in the developing chick embryo relating its expression to the onset of myogenic differentiation and cellular fate within the craniofacial mesoderm. We show that Tbx1 is expressed before capsulin, the first known marker of branchial arch 1 and 2 muscles. We also show that, as in the mouse, Tbx1 is expressed in endothelial cells, another mesodermal derivative, and, therefore, Tbx1 alone cannot specify the myogenic lineage. In addition, Tbx1 expression was identified in both chick and mouse limb myogenic cells, initially being restricted to the dorsal muscle mass, but in contrast, to the head, here Tbx1 is expressed after the onset of myogenic commitment. Functional studies revealed that loss of Tbx1 function reduces the number of myocytes in the head and limb, whereas increasing Tbx1 activity has the converse effect. Finally, analysis of the Tbx1‐mesoderm‐specific knockout mouse demonstrated the cell autonomous requirement for Tbx1 during myocyte development in the cranial mesoderm. Developmental Dynamics 236:353–363, 2007.

Sonic Hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.

Wnt/Lef1 signaling acts via Pitx2 to regulate somite myogenesis

Wnt signaling has been implicated in somite, limb, and branchial arch myogenesis but the mechanisms and roles are not clear. We now show that Wnt signaling via Lef1 acts to regulate the number of premyogenic cells in somites but does not regulate myogenic initiation in the limb bud or maintenance in the first or second branchial arch. We have also analysed the function and regulation of a putative downstream transcriptional target of canonical Wnt signaling, Pitx2. We show that loss-of-function of Pitx2 decreases the number of myogenic cells in the somite, whereas overexpression increases myocyte number particularly in the epaxial region of the myotome. Increased numbers of mitotic cells were observed following overexpression of Pitx2 or an activated form of Lef1, suggesting an effect on cell proliferation. In addition, we show that Pitx2 expression is regulated by canonical Wnt signaling in the epaxial somite and second branchial arch, but not in the limb or the first branchial arch. These results suggest that Wnt/Lef1 signaling regulates epaxial myogenesis via Pitx2 but that this link is uncoupled in other regions of the body, emphasizing the unique molecular networks that control the development of various muscles in vertebrates.

Cell death after dorsal root injury

Flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate nick end-labelling (TUNEL) immunohistochemistry have been used to assess cell death in the dorsal root ganglia (DRG) or spinal cord 1, 2 or 14 days after multiple lumbar dorsal root rhizotomy or dorsal root avulsion injury in adult rats. Neither injury induced significant cell death in the DRG compared to sham-operated or naïve animals at any time point. In the spinal cord, a significant increase in death was seen at 1-2 days, but not 14 days, post injury by both methods. TUNEL staining revealed that more apoptotic cells were present in the dorsal columns and dorsal horn of avulsion animals compared to rhizotomised animals. This suggests that avulsion injury, which can often partially damage the spinal cord, has more severe effects on cell survival than rhizotomy, a surgical lesion which does not affect the spinal cord. The location of TUNEL positive cells suggests that both neuronal and non-neuronal cells are dying.

Riluzole promotes cell survival and neurite outgrowth in rat sensory neurones in vitro

This study explored the effects of riluzole administration on cell survival and neurite growth in adult and neonatal rat dorsal root ganglion (DRG) neurones in vitro. Neuronal survival was assessed by comparing numbers of remaining neurones in vehicle‐ and riluzole‐treated cultures. A single dose of 0.1 µm riluzole was sufficient to promote neuronal survival in neonatal DRG cultures, whereas repeated riluzole administration was necessary in adult cultures. However, a single administration of riluzole was sufficient to induce neuritogenesis, promote neurite branching and enhance neurite outgrowth in both neonatal and adult DRG cultures. The effects of a single dose of riluzole on adult DRG neurones after peripheral nerve or dorsal root injury were also studied in vitro at 48 h. For both types of injury, riluzole enhanced neurite outgrowth in terms of number, length and branch pattern significantly more on the injured side as compared with the contralateral side. No effect was seen on cell survival. The results suggest that, in addition to its cell survival effects, riluzole has novel growth‐promoting effects on sensory neurones in vitro and that riluzole may offer a new way to promote sensory afferent regeneration following peripheral injury.

Bmi1 is expressed in postnatal myogenic satellite cells, controls their maintenance and plays an essential role in repeated muscle regeneration.

Satellite cells are the resident stem cell population of the adult mammalian skeletal muscle and they play a crucial role in its homeostasis and in its regenerative capacity after injury. We show here that the Polycomb group (PcG) gene Bmi1 is expressed in both the Pax7 positive (+)/Myf5 negative (−) stem cell population as well as the Pax7+/Myf5+ committed myogenic progenitor population. Depletion of Pax7+/Myf5− satellite cells with reciprocal increase in Pax7+/Myf5+ as well as MyoD positive (+) cells is seen in Bmi1−/− mice leading to reduced postnatal muscle fiber size and impaired regeneration upon injury. Bmi1−/− satellite cells have a reduced proliferative capacity and fail to re-enter the cell cycle when stimulated by high serum conditions in vitro, in keeping with a cell intrinsic defect. Thus, both the in vivo and in vitro results suggest that Bmi1 plays a crucial role in the maintenance of the stem cell pool in postnatal skeletal muscle and is essential for efficient muscle regeneration after injury especially after repeated muscle injury.